Methods for treating juvenile arthritis with anti-bile salt-stimulated lipase (bssl) antibodies

ABSTRACT

It provides methods and pharmaceutical compositions comprising antagonists to the protein Bile Salt-Stimulated Lipase (BSSL) for the prevention, prophylaxis and treatment of inflammatory diseases, such as rheumatoid arthritis. It further relates to pharmaceutical compositions comprising BSSL antagonists and their use in methods for the prevention, prophylaxis and treatment of inflammatory diseases, such as rheumatoid arthritis. Suitable BSSL antagonists to be used according to the invention are BSSL antibodies.

RELATED APPLICATIONS

This application is a Continuation Application of U.S. application Ser.No. 14/868,129, filed Sep. 28, 2015, which is a Continuation Applicationof U.S. application Ser. No. 14/067,495, filed Oct.30, 2013, now U.S.Pat. No. 9168299, which is a Continuation Application of U.S.application Ser. No. 13/262,805, filed Jan. 9, 2012, now U.S. Pat. No.8597650, which is a U.S. National Phase of International Application No.PCT/SE2010/050377, filed Apr. 6, 2010, designating the U.S., andpublished as WO 2010/117325 on Oct. 14, 2010, which claims the benefitof U.S. Provisional Application No. 61/254,221 filed Oct. 23, 2009, andSwedish Patent Application No. 0950228-7 filed Apr. 8, 2009.

SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is herebyincorporated by reference in accordance with 37 U.S.C. § 1.52(e). Thename of the ASCII text file for the Sequence Listing isSeqList-BRB007-001C3.txt, the date of creation of the ASCII text file isMay 29, 2020, and the size of the ASCII text file is 91 KB.

TECHNICAL FIELD

The invention provides methods and pharmaceutical compositionscomprising antagonists to the protein Bile Salt-Stimulated Lipase (BSSL)for the prevention, prophylaxis and treatment of inflammatory diseases,such as rheumatoid arthritis. The invention further relates topharmaceutical compositions comprising BSSL antagonists and their use inmethods for the prevention, prophylaxis and treatment of inflammatorydiseases, such as rheumatoid arthritis.

BACKGROUND Inflammatory Diseases—Rheumatoid Arthritis

Inflammation, a reaction of the body to injury or to infectious,allergic, or chemical irritation can lead to a varity of inflammatorydiseases or disorders such as inflammation associated with allergy,inflammation related to the production of nitric oxide, inflammationrelated to the skin, abdomen, peripheral or central nervous system, eyeor tear glands, ear, nose, mouth, lung, heart, liver, pancreas, thyroid,adipose tissue, kidney, joints or blood vessels, or inflammation relatedto infection, trauma or autoimmunity.

Rheumatoid arthritis (RA) is a chronic, inflammatory, systemicautoimmune disease that affects about 1% of the general population inWestern societies (Gabriel 2001). The disease process results inprogressive destruction of joint cartilage and bone. This destructionresults from immune responses and non-antigen-specific innateinflammatory processes. The disease is characterized by mono- orpolyarticular joint inflammation with massive accumulation ofneutrophils in the synovial fluid and tissue. The synovial neutrophilscontribute to cartilage destruction by releasing proteases andgenerating oxi-dants and it is becoming more and more evident thatinhibiting neutrophil infiltration into inflamed joints could be anapproach to prevent progression of the disease (Hallett 2008).

Current therapies for RA include non-steroid anti-inflammatory drugs(NSAIDs) for pain treatment, disease-modifying antirheumatic drugs(DMARDs) and biological agents that target specific proinflammatorycytokines, or cell surface receptors of various cell types.

There remains a need, however, for alternative pharmaceutical treatmentsof inflammatory diseases, especially chronic inflammatory diseases.Consequently there is a need to identify new unique targets involved ininflammatory signaling and processes, which can be used as the basis fordevelopment of new innovative therapeutic agents for the treatment,prophylaxis and prevention of inflammatory diseases.

Bile Salt-Stimulated Lipase

The bile salt-stimulated lipase (BSSL) also designated carboxyl esterlipase (CEL) or bile salt-dependent lipase (BSDL) is a lipolytic enzymeexpressed in the exocrine pancreas and secreted into the intestinallumen in all species so far investigated. In some species, including thehuman, BSSL is also expressed by the lactating mammary gland andsecreted with the milk. BSSL has broad substrate specificity withcapacity to hydrolyze a variety of different substrates, e.g.cholesteryl esters, tri-, di-, and monoacylglycerols, fat-solublevitamin esters, phospholipids, galactolipids and ceramides (Hui andHowles 2002). The physiological function of BSSL was originally thoughtto be confined to the small intestine and hydrolysis of dietary fat(Hernell et al. 1997). The high abundance of BSSL in pancreatic juice(up to 5% of total protein content) and the ability of BSSL to hydrolyzea broad spectrum of lipids have led researchers to suggest a variety offunctions for BSSL in lipid digestion and absorption. BSSL has a keyrole in the absorption of cholesteryl esters (Fält et0 al. 2002),verified in mice lacking the BSSL (CEL gene) (Howles et al. 1996). Whilethis is considered its main function in the human adult it is likely tocontribute also to triglyceride digestion and absorption in the newborninfant (Lindquist and Hernell 2010).

BSSL was found to be present in low, but significant levels in serum ofhealthy individuals (Bläckberg et al. 1985) and current research hasimplicated that BSSL is involved in lipoprotein metabolism andmodulation of atherosclerosis (Hui and Howles 2002). The potentialfunction, or even the question if elevated levels of circulating BSSL isa risk factor for, or protects against atherosclerosis is not clear. Asurprisingly strong positive association between BSSL, assayed ascholesterol esterase activity, and total—as well as low-densitylipoprotein (LDL)-cholesterol levels in serum was first reported (Huiand Howles 2002). BSSL was then shown to be associated with smoothmuscle cells (SMCs) within atherosclerotic plaques and to inducevascular SMC proliferation in vitro (Auge et al. 2003). A study, usingtransgenic mice, demonstrated that macrophage expression of BSSL ispro-atherogenic, favouring cholesteryl ester accumulation and foam cellformation (Kodvawala et al. 2005). Judged by these studies BSSL would bea risk factor for atherosclerosis. On the other hand, BSSL reduceslysophosphatidylcholine content in oxidized LDL, thereby reducingaccumulation of oxidized LDL in macrophages (Hui and Howles 2002), andit has been suggested to play a physiological role in hepatic selectiveuptake and metabolism of high density lipoprotein cholesteryl esters bydirect and indirect interactions with the scavenger receptor BI pathway(Camarota et al. 2004), which implicates that BSSL in serum protectsagainst atherosclerosis.

The BSSL Protein

The human BSSL protein (encoded by the CEL gene) is a single-chainglycoprotein of 722 amino acids (Nilsson et al. 1990). The enzyme issynthesised as a precursor of 742 amino acids with a signal peptide of20 amino acids. Two bile salt-binding sites regulating the activity ofthe enzyme and the resistance to proteases have been postulated (Hui1996) as well as a sphingolipid binding domain (SBD) (Aubert-Jousset etal. 2004).

Schematically the enzyme can be divided into two parts:

i) The N-terminal domain with a striking homology toacetylcholinesterase and some other esterases. In this part the proposedcatalytic triad (Ser194 (included in the motif GESAG), Asp320 andHis435) are found as well as a N-glycosylation site, Asn187, aheparin-binding site (postulated to be located at position 1-100) andthe two intra chain disulfide bridges (Cys64-Cys80 and Cys 246-Cys257).The heparin binding ability has been found to be located in the part ofthe molecule consisting of amino acids 1-445 (Spilburg et al. 1995) andthe heparin binding domain may, in fact, be a three-dimensionalstructure composed of different sequences. The heparin bindingproperties of BSSL is thought to be important for interactions with cellmembranes, exemplified by intestinal cell membranes (Fält 2002).

ii) The C-terminal part (encoded by exon 11) with a variable number oftandem repeats (VNTR) -region containing similar but not identicalrepeats (11 amino acids). The most common human form contains 16, butthere is a variation in number of repeats both between individuals andalleles (Lindquist et al. 2002). The repeats are followed by an extratail of 11 amino acids (this tail is longer in the corresponding rat andmouse enzyme). The repeats are proline-rich and the presence of asparticacid in every repeating unit and glutamic acid in some, render thisregion highly acidic and contributes to the low iso-electric point ofthe protein. The number of proline-rich repeats has been reported tovary extensively between species, typically ranging from three in mouseand the cow, four in the rat to 16 in humans and 39 in the gorilla (Huiand Howles 2002; Madeyski et al. 1999). This diversity in number ofrepeated units can explain the observed size differences of the proteinbetween species; the mouse BSSL is a 74kDa protein while the human BSSL,which is extensively glycosylated across the repeated region, has anapparent molecular mass of 120-140 kDa; the repeats carry most of the15-35% carbohydrate of the protein. The varying apparent molecular masscan be explained both by the number of repeats and differences inglycosylation (Lindquist et al. 2002). It has been shown by analysingthe isolated C-terminal part of human milk BSSL (amino acids 528-722)that probably only 10 out of 16 repeats in human milk BSSL areO-glycosylated (Wang et al. 1995).

It has been suggested that the repeats may have a functional role inprotecting BSSL from proteolytic degradation and that theirO-glycosylation is important for secretion of the enzyme (Bruneau et al.1997). The oligosaccharides in the C-terminal region contain Lewis x andLewis b and less Lewis a antigenic structures. Owing to thoseblood-group-related antigenic determinants, the C-terminal region ofBSSL may have an adhesive function in cell-cell interactions, asillustrated by its antimicrobial effects (Naarding et al. 2006;Ruvoën-Clouet et al. 2006). On the other hand, the repeated region maybe less important for catalytic activity, activation by bile salts andheparin binding (Hui 1996).

The C-tail has also been suggested to be an important structural part bybinding to a lectin-like receptor (LOX-1) on the surface of intestinalendothelium cells (Fayard et al. 2003). The heparin binding site(s)forms the other binding part, and these binding sites have a pivotalrole in the mechanism of action for BSSL in different cellularenvironments and cell stages.

Vascular BSSL

Comparison of BSSL VNTR genotype and serum lipid phenotype revealed anassociation between the number of repeats and serum cholesterol profile(Bengtsson-Ellmark et al. 2004). While it is possible that the repeatpolymorphism is merely a genetic marker for lipid profile, it is alsopossible that it has functional role in determining plasma lipidcomposition.

A wider role for BSSL in lipid metabolism is implicated by the presenceof BSSL in human plasma and aortic tissue. The source of circulatingBSSL has been discussed extensively. Human macrophages and endothelialcells were shown to synthesize and secrete the enzyme (Hui and Howles2002). Conversely, in another study BSSL within atherosclerotic lesionswas associated with smooth muscle cells (SMCs) but not with activatedmacrophages or endothelial cells (Augé et al. 2003). In yet anotherstudy, BSSL injected into rat intestinal loops was advocated to beinternalized by enterocytes, transferred through the cells and releasedinto the circulation (Bruneau et al. 2003). Based on these data it wasproposed that circulating BSSL originates from the pancreas. However, ithas been further shown that neither does the BSSL serum level increaseafter a meal of breast milk, nor does it differ between breastfed andformula fed human infants, although in the newborn breast milk is themajor source of BSSL, while it is absent from infant formula (Bläckberget al. 1985; Shamir et al. 2003).

An association of BSSL with apolipoprotein B-containing lipoproteins inhuman plasma has been reported (Bruneau et al. 2003), which togetherwith the observation that BSSL is present in the human aorta and has theability to modify low density lipoprotein (LDL) and high densitylipoprotein (HDL) composition and reduce the atherogenicity of oxidizedLDL (oxLDL) by decreasing their lysophosphatidylcholine (lysoPC) content(Shamir et al. 1996), invoked a potential new role for BSSL as aprotective factor in the development of atherosclerosis. LysoPC is amajor phospholipid component in oxLDL and is generated by oxidation andfragmentation of polyunsaturated fatty acids esterified to the sn-2position of the PC molecule, followed by hydrolysis of the shortenedfatty acyl residue by LDL-associated phosolipase A2 (PLA2) and BSSL.Although lysoPC constitutes only 1-5% of total PC in non-oxLDL,oxidative modification of LDL can raise this proportion to as high as40-50%. LysoPC acts as a chemoattractant for monocytes, induces monocyteadhesion to the vascular endothelium and promotes macrophageproliferation, which eventually leads to foam cell formation. Due to itseffects on lysoPC, it has been suggested that BSSL may interact withcholesterol and oxidized lipoproteins to modulate the progression ofatherosclerosis (Hui and Howles 2002).

However, the fact that BSSL is found and accumulated in atheroscleroticlesions, and the fact that monocytes as well as macrophages (or SMChaving a macrophage phenotype) express and secrete BSSL, indicate thatthese cells may be a possible source of the accumulated BSSL. Themechanism behind a pathophysiological role of BSSL in macrophages issuggested to be the function of BSSL as a ceramidase (Hui and Howles2002) by its reduction of ceramide and lysophosphatidylcholine levelsleading to increased cholesteryl ester accumulation in response toatherogenic lipoproteins resulting in increased atherosclerosis lesionsize in vivo. This is in line with the study by Kodvawala et al. (2005),who by using in vivo models showed that BSSL expression in macrophagespromotes cholesteryl ester synthesis and accumulation in response tomodified LDL and increases atherosclerosis lesions in apoE deficientmice.

The Response to Retention Hypothesis of Atherosclerosis

Many of the processes implicated in the early stages of atherogenesisincluding endothelial damage, lipoprotein oxidation and macrophage andVSMC (vascular smooth muscle cells) proliferation are individually notsufficient to lead to lesion development. The response-to-retentionhypothesis suggests that subendothelial retention of atherogeniclipoproteins is the trigger for all of these processes which are in factnormal physiological responses to the accumulation of lipids.

While the major determinant of initial retention of LDL is likely to bethe proteoglycan composition within the subendothelial space, BSSL mayfacilitate and enforce retention once the lesion has started to form byacting as a molecular bridge between the subendothelial proteoglycansand lipoproteins (WO 2005/095986). The BSSL that is bound to thecomponents of the extracellular matrix can act as bridging molecules inthe retention of LDL, as suggested for Lipoprotein lipase (LPL)(Pentikainen et al. 2002).

BSSL in Platelets

Recently BSSL was found to be stored in blood platelets and releasedupon platelet activation (Panicot-Dubois et al. 2007). Moreover, BSSLwas shown to induce calcium mobilization in platelets and to enhancethrombin-mediated platelet aggregation and spreading.

In a mouse thrombosis model (laser-induced injury), BSSL accumulated inarterial thrombi in vivo—at sites of vessel wall injury. When CXCchemokine receptor 4 (CXCR4) was antagonized, the accumulation of BSSLwas inhibited and thrombus size was reduced. In BSSL knockout mice(BSSL-KO) tail bleeding times were increased in comparison with those ofwild-type mice. These data suggest that BSSL modulates thrombusformation by interacting with CXCR4 on platelets.

CXCR4 belongs to the G-protein-coupled receptor (GPCR) gene family, andupon acti-vation CXCR4 induces downstream signaling by several differentpathways; e.g. CXCR4 binding of the chemokine ligand SDF-1 activatesG-protein mediated signaling and induces cellular chemotactic responses(Clemetson et al. 2000). CXCR4 is also known to interact with HIV-1 andto act as a co-receptor for entry of the virus into cells. The bindingof HIV-1 to CXCR4 is mediated via a domain denoted the V3 loop presenton HIV-1 gp120. The BSSL protein contains a region that is structurallyrelated to the V3-loop of gp120. This region, called the V3-like loopdomain (amino acids 361-393) (Aubert-Jousset et al. 2004) was proposedto mediate the binding of BSSL to CXCR4 on platelets.

In summary, there are both confusing and conflicting result regardingthe source and function of BSSL in plasma and aortic tissue.

EP 1840573 reports on differences in gene expression pattern between NOD(non-obese diabetic) mice positive or negative for insulinautoantibodies. 125 differentially expressed genes were identified, oneof them being the CEL gene encoding BSSL. The differentially expressedgenes are identified as having utility in early diagnosis of apre-inflammatory state of autoimmune diseases, such as type I diabetes.

The differentialy expressed genes are further suggested to be targetsfor the treatment of autoimmune diseases having a pre-inflammatoryphase. It is well known in the art that expression of numerous genes isaltered as a consequence of the development of a specific disease, asdemonstrated in EP 1840573. However, all such differentially expressedgenes can not be considered to be the cause of the development of thedisease. On the contrary the identification of the causative gene(s), ifat all existing, requires further complicated investigations. EP1840573, even if identifying BSSL as potential marker for inflammatorydisease, fails to identify BSSL as a cause for the development ofinflammatory disease.

SUMMARY

The present invention is based on the surprising discovery that BSSL hasa role in inflammatory processes and that inhibition or elimination ofBSSL protects from development of chronic arthritis in animal models.

The present invention is based on the demonstration that BSSL deficientmice are protected from development of inflammatory disease, exemplifiedby collagen-induced arthritis (CIA). Consequently antagonists to humanBSSL are potentially useful for prevention, prophylaxis and/or treatmentof inflammatory diseases. Suitable antagonists to BSSL are agents thatreduce the activity, amount and/or expression of BSSL. Preferred

BSSL antagonists which can be used according to the present inventionare antibodies and antibody fragments specifically binding to humanBSSL, as well as RNAi and antisense polynucleotides comprising sequencescomplementary to a polynucleotide sequences encoding human BSSL. Mostpreferably the BSSL antoginists to be used according to the inventionare monoclonal BSSL antibodies.

Accordingly, one aspect of the present invention provides a method forthe prevention, prophylaxis and/or treatment of an inflammatory diseasecomprising administering a pharmaceutical effective amount of anantibody or an antibody fragment specifically binding to human BSSL to asubject in need of such treatment.

Another aspect of the present invention provides a pharmaceuticalcomposition comprising an antibody or an antibody fragment specificallybinding to human BSSL, and a pharmaceutically acceptable carrier orexcipient for use in the prevention, prophylaxis and/or treatment of aninflammatory disease.

Yet another aspect of the present invention provides use of an antibodyor an antibody fragment specifically binding to human BSSL in themanufacture of a pharmaceutical composition for the prevention,prophylaxis and/or treatment of an inflammatory disease.

Another aspect of the present invention provides a method for theprevention, prophylaxis and/or treatment of an inflammatory diseasecomprising administering a pharmaceutical effective amount of an RNAimolecule or an antisense polynucleotide comprising a sequencecomplementary to a part of a polynucleotide sequence encoding human BSSLor a sequence complementary thereto to a subject in need of suchtreatment.

Another aspect of the present invention provides a pharmaceuticalcomposition comprising an RNAi molecule or an antisense polynucleotidecomprising a sequence complementary to a part of a polynucleotidesequence encoding human or a sequence complementary thereto, and apharmaceutically acceptable carrier or excipient for use in theprevention, prophylaxis and/or treatment of an inflammatory disease.

Yet another aspect of the present invention provides use of an RNAimolecule or an antisense polynucleotide comprising a sequencecomplementary to a part of a polynucleotide sequence encoding human BSSLor a sequence complementary thereto in the manufacture of apharmaceutical composition for the prevention, prophylaxis and/ortreatment of an inflammatory disease.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Detection of BSSL mRNA in human liver.

Total RNA, isolated in duplicate from liver biopsies of fourindividuals, was reverse-transcribed and amplified using BSSL-specificoligonucleotide primers. The PCR products were resolved by 1.8% agarosegel electrophoresis and stained with ethidium bromide. A PCR product ofthe expected size (327 nt) was amplified from all samples; patient 1(lanes 1 and 2); patient 2 (lanes 3 and 4); patient 3 (lane 5 and 6);patient 4 (lanes 7 and 8). cDNA synthesized from RNA isolated from humanmilk was used as a positive control (lane 9). The O'GeneRuler™ 50-bp DNAladder (Fermentas, Ontario, Canada) was used as a molecular size marker(lane 10).

FIG. 2. Western blot.

Affinity-purified protein extracts derived from two human liver samples(patient no. 3 and no. 4), were separated by SDS-PAGE (10%), transferredto PVDF membranes, and probed with a polyclonal anti-human BSSLantibody. Patient 3, lane 1; patient 4, lane 2. Protein extracts fromhuman milk, lane 3; human pancreas, lane 4; and BSSL purified from humanmilk, lane 5, were used as positive controls.

FIG. 3. Histology, oil red O staining, and BSSL localization in humanliver sections.

Liver tissue sections (8-μm cryosections) obtained from two patients[patient 1 (A-C); patient 4 (D-F)] were stained with hematoxylin andeosin (A, D), oil red O (B, E), and immunohistochemistry with polyclonalanti-BSSL antibodies (C, F).

FIGS. 4A and 4B. Double immunofluorescence stainings against BSSL andimmune cell markers.

BSSL co-localizes with CD15 but not with CD68-expressing cells in humanliver. Double-immunofluorescence staining of liver sections (8 μm)obtained from two patients [patient 1 (FIG. 4A) and patient 4 (FIG. 4B)]using a rabbit polyclonal anti-BSSL antibody and mouse monoclonalanti-CD68 or anti-CD15 antibodies. A yellow color appeared in the mergedpicture in both panels when anti-BSSL and anti-CD15 antibodies were usedtogether, seen as bright staining in these black and white figures,indicating co-localization.

FIG. 5. BSSL localizes to circulating CD15-positive granulocytes.

Human leukocytes were harvested from blood of healthy volunteers,permeabilized and stained by double immunofluorescence using rabbitpolyclonal anti-BSSL (A) and mouse monoclonal anti-CD15 (B) antibodies.Cell nuclei were counterstained with DAPI (C). A yellow color appearedin the merged picture (D), seen as bright staining in this black andwhite figure, indicating co-localization.

FIG. 6. Subcellular localization of BSSL in circulating granulocytes.

Human leukocytes were harvested from blood of healthy volunteers andstained by double immunofluorescence using rabbit polyclonal anti-BSSLand mouse monoclonal anti-CD15 antibodies. To distinguish betweenextracellular and intracellular localization, cells were eitherpermeabilized (upper panel) or not (bottom panel) before antibodies wereapplied. A yellow color appeared in the merged picture in the upperpanel, seen as bright staining in these black and white figures,indicating co-localization.

FIG. 7. Western blot analysis.

Affinity-purified protein extracts derived from human mononuclear bloodcells (lanes 1 and 2) or polynuclear granulocytes (lanes 3-5) wereseparated by SDS-PAGE (10%), transferred to PVDF membranes, and probedwith a polyclonal anti-human BSSL antibody. Protein extracts from humanmilk (lane 6) and human pancreas (lane 7) were used as positivecontrols.

FIG. 8. Detection of BSSL mRNA in human blood cells.

Total RNA isolated from mononuclear blood cells and polynucleargranulocytes from two healthy individuals was reverse-transcribed andamplified using BSSL-specific oligonucleotide primers. The PCR productswere resolved by 1.8% agarose gel electrophoresis and stained withethidium bromide. A PCR product of the expected size (327 nt) wasamplified from all samples. Mononuclear blood cells (lanes 1 and 2);polynuclear granulocytes (lanes 3 and 4). Negative controls (omitting RTfrom the cDNA synthesis reaction) are shown in lanes 5-8. TheO'GeneRuler™ 50-bp DNA ladder (Fermentas) was used as a molecular sizemarker (lane 9).

FIG. 9. Immunolocalization of in human atherosclerotic plaque.

Immunohistochemistry was performed on formalin-fixed, paraffin-embeddedtissue sections obtained from atherosclerotic carotid arteries using arabbit polyclonal BSSL-peptide (amino acid 328-341) antibody (A) and (C)or rabbit pre-immune serum (B) and (D), as negative control. Mayer'shematoxylin was used for counterstaining. The figure shows data from twopatients (A, B are sections from patient 1; C, D are sections frompatient 2).

FIGS. 10A, 10B and 10C. Mean arthritis score in CIA mouse model.

Arthritis was followed for 57 days by scoring 2-3 times a week.deficient mice developed highly significantly lower disease scorecompared to wt controls. There was a profound difference in diseasesusceptibility between the sexes. Only few female mice developedarthritis and those who did had low score. (FIG. 10A) all mice; (FIG.10B) males; (FIG. 10C) females.

FIGS. 11A and 11B. Incidence and severity in CIA mouse model.

The BSSL deficient mice developed arthritis with reduced incidence andalso lower severity compared to their wt littermates. Incidence is shownas percent of all mice (FIG. 11A) and severity is shown as meanarthritis score of sick mice only (FIG. 11B).

FIGS. 12A and 12B. Serum concentration of anti-CII antibodies in CIAmouse model.

Analysis of anti-collagen II (anti-CII antibody) concentration in serumwithdrawn at day 30 (FIG. 12A) and day 57 (FIG. 12B) revealed nodifferences in response between BSSL deficient (black bars) and BSSL wtmice (white bars) in neither of the IgG isotypes (represented by totalIgG in the figure), nor IgM.

FIG. 13. Cartilage degradation in CIA mouse model.

The concentration of cartilage oligomeric matrix protein (COMP) in serumat day 57 was measured by ELISA as a marker for cartilage degradation.The level of COMP was significantly lower in BSSL deficient males (blackbar) compared to wt male controls (white bar). In females there was nodifference.

FIGS. 14A, 14B, and 14C. Mean arthritis score, arthritis severity andincidence in CIA mouse model.

Arthritis was followed for 48 days by scoring 2-3 times a week.deficient mice showed a significantly lower disease score compared toBSSL wt mice (FIG. 14A) and (FIG. 14C), which was also reflected by alower incidence of arthritis (FIG. 14B). BSSL heterozygous mice wereless prone to develop disease as compared to BSSL wt mice but not asresistant as homozygous BSSL deficient mice. (FIG. 14A) and (FIG. 14B)all mice; (FIG. 14C) sick mice only. * represents p<0.05 and **represents p<0.01.

FIG. 15. Cartilage degradation in CIA mouse model.

The concentration of COMP in serum at day 48 was measured by ELISA as amarker for cartilage degradation. The level of COMP was significantlylower in BSSL deficient mice (black bar) compared to BSSL wt controls(white bar). The serum concentration of COMP in BSSL heterozogous mice(hatched bar) was found to be intermediate in relation to theconcentration in deficient and BSSL wt mice. * represents p<0.05.

FIG. 16. Anti-collagen type II response (IgG) in plasma.

Analysis of anti-CII antibody levels at day 33 and day 48 presented asrelative values compared to a standard of pooled serum. There was nosignificant difference in IgG response between any of the BSSLgenotypes.

FIG. 17. Arthritis severity after anti-BSSL injections compared tocontrol.

Rats injected with either 1 mg/kg or 5 mg/kg anti-BSSL showedsignificantly decreased disease severity. * represents p<0.05 and **represents p<0.01. Incidence was 100% for all groups.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention is based on the discovery that BSSL has a role ininflammatory processes and that inhibition or elimination of BSSLprotects from development of chronic arthritis in animal models. It isdemonstrated that the BSSL protein is present in inflammatory cells andinflamed tissue. BSSL-deficient mice (BSSL-KO) developedcollagen-induced arthritis (CIA) with significantly reduced diseaseseverity and less incidence compared to wild-type controls. Injection ofanti-BSSL antibodies significantly reduced disease severity ofpristane-induced arthritis in rats.

The invention provides BSSL antagonists for the prevention and/ortreatment of inflammatory diseases. Preferably, the BSSL antagonist canbe an antibody or an antibody fragment specifically binding to humanBSSL, or an RNAi molecule or an antisense polynucleotide comprising asequence complementary to a part of a polynucleotide sequence encodinghuman BSSL.

Inflammatory Diseases

Inflammatory diseases that can be prevented and/or treated according tothe invention are diseases selected from, but not limited to;

inflammatory diseases of the respiratory tract including: asthma,including bronchial, allergic, intrinsic, extrinsic, exercise-induced,drug-induced (including aspirin and NSAID-induced) and dust-inducedasthma, both intermittent and persistent and of all severities, andother causes of airway hyper-responsiveness; chronic obstructivepulmonary disease (COPD); bronchitis, including infectious andeosinophilic bronchitis; emphysema; bronchiectasis; cystic fibrosis;sarcoidosis; farmer's lung and related diseases; hypersensitivitypneumonitis; lung fibrosis, including cryptogenic fibrosing alveolitis,idiopathic interstitial pneumonias, fibrosis complicatinganti-neoplastic therapy and chronic infection, including tuberculosisand aspergillosis and other fungal infections; complications of lungtransplantation; vasculitic and thrombotic disorders of the lungvasculature, and pulmonary hypertension; antitussive activity includingtreatment of chronic cough associated with inflammatory and secretoryconditions of the airways, and iatrogenic cough; acute and chronicrhinitis including rhinitis medicamentosa, and vasomotor rhinitis;perennial and seasonal allergic rhinitis including rhinitis nervosa (hayfever); nasal polyposis; acute viral infection including the commoncold, and infection due to respiratory syncytial virus, influenza,coronavirus (including SARS) and adenovirus;

inflammatory diseases of bone and joints includingosteoarthritis/osteoarthrosis, both primary and secondary to, forexample, congenital hip dysplasia; cervical and lumbar spondylitis, andlow back and neck pain; rheumatoid arthritis and Still's disease;seronegative spondyloarthropathies including ankylosing spondylitis,psoriatic arthritis, reactive arthritis and undifferentiatedspondarthropathy; septic arthritis and other infection-relatedarthopathies and bone disorders such as tuberculosis, including Potts'disease and Poncet's syndrome; acute and chronic crystal-inducedsynovitis including urate gout, calcium pyrophosphate depositiondisease, and calcium apatite related tendon, bursal and synovialinflammation; Behcet's disease; primary and secondary Sjogren'ssyndrome; systemic sclerosis and limited scleroderma; systemic lupuserythematosus, mixed connective tissue disease, and undifferentiatedconnective tissue disease; inflammatory myopathies includingdermatomyositits and polymyositis; polymalgia rheumatica; juvenilearthritis including idiopathic inflammatory arthritides of whateverjoint distribution and associated syndromes, and rheumatic fever and itssystemic complications; vasculitides including giant cell arteritis,Takayasu's arteritis, Churg-Strauss syndrome, polyarteritis nodosa,microscopic polyarteritis, and vasculitides associated with viralinfection, hypersensitivity reactions, cryoglobulins, and paraproteins;low back pain; Familial Mediterranean fever, Muckle-Wells syndrome, andFamilial Hibernian Fever, Kikuchi disease; drug-induced arthalgias,tendonititides, and myopathies;

inflammatory diseases related to connective tissue remodelling ormusculoskeletal disorders due to injury (for example sports injury) ordisease including arthritides (for example rheumatoid arthritis,osteoarthritis, gout or crystal arthropathy), other joint disease (suchas intervertebral disc degeneration or temporomandibular jointdegeneration), bone remodelling disease (such as osteoporosis, Paget'sdisease or osteonecrosis), polychondritits, scleroderma, mixedconnective tissue disorder, spondyloarthropathies or periodontal disease(such as periodontitis);

inflammatory cardiovascular diseases including atherosclerosis,affecting the coronary and peripheral circulation; pericarditis;myocarditis, inflammatory and auto-immune cardiomyopathies includingmyocardial sarcoid; ischaemic reperfusion injuries;

endocarditis, valvulitis, and aortitis including infective (for examplesyphilitic); vasculitides; disorders of the proximal and peripheralveins including phlebitis and thrombosis, including deep vein thrombosisand complications of varicose veins;

inflammatory disease of the skin including psoriasis, atopic dermatitis,contact dermatitis or other eczematous dermatoses, and delayed-typehypersensitivity reactions; phyto-and photodermatitis; seborrhoeicdermatitis, dermatitis herpetiformis, lichen planus, lichen sclerosus etatrophica, pyoderma gangrenosum, skin sarcoid, discoid lupuserythematosus, pemphigus, pemphigoid, epidermolysis bullosa, urticaria,angioedema, vasculitides, toxic erythemas, cutaneous eosinophilias,alopecia areata, male-pattern baldness, Sweet's syndrome,Weber-Christian syndrome, erythema multiforme; cellulitis, bothinfective and non-infective; panniculitis; cutaneous lymphomas,non-melanoma skin cancer and other dysplastic lesions; drug-induceddisorders including fixed drug eruptions;

inflammatory disease of the eyes including blepharitis; conjunctivitis,including perennial and vernal allergic conjunctivitis; iritis; anteriorand posterior uveitis; choroiditis; autoimmune; degenerative orinflammatory disorders affecting the retina; ophthalmitis includingsympathetic ophthalmitis; sarcoidosis; infections including viral,fungal, and bacterial;

inflammatory diseases of the gastrointestinal tract including glossitis,gingivitis, periodontitis; oesophagitis, including gastroesophagealreflux disease; eosinophilic gastro-enteritis, mastocytosis, celiacdisease, Crohn's disease, colitis, ulcerative colitis, proctitis,pruritis ani, irritable bowel disorder, irritable bowel syndrome,

abdominal inflammatory diseases including hepatitis, includingautoimmune, alcoholic and viral; fibrosis and cirrhosis of the liver;cholecystitis; pancreatitis, both acute and chronic;

genito-urinary tract inflammatory diseases including nephritis includinginterstitial and glomerulonephritis; nephrotic syndrome; cystitisincluding acute and chronic (interstitial) cystitis and Hunner's ulcer;acute and chronic urethritis, prostatitis, epididymitis, oophoritis andsalpingitis; vulvovaginitis; Peyronie's disease; erectile dysfunction(both male and female);

allograft rejection including acute and chronic following, for example,transplantation of kidney, heart, liver, lung, bone marrow, skin orcornea or following blood transfusion; or chronic graft versus hostdisease;

inflammatory central nervous system diseases including Alzheimer'sdisease and other dementing disorders including Creutzfeldt-Jakobdisease and New varaint Creutzfeldt-Jakob disease; amyloidosis; multiplesclerosis and other demyelinating syndromes; cerebral atherosclerosisand vasculitis; temporal arteritis; myasthenia gravis; acute and chronicpain (acute, intermittent or persistent, whether of central orperipheral origin) including visceral pain, headache, migraine,trigeminal neuralgia, atypical facial pain, joint and bone pain, painarising from cancer and tumor invasion, neuropathic pain syndromesincluding diabetic, post-herpetic, and HIV-associated neuropathies;neurosarcoidosis; central and peripheral nervous system complications ofmalignant, infectious or autoimmune processes; and

other auto-immune and allergic disorders including Hashimoto'sthyroiditis, Graves' disease, Addison's disease, diabetes mellitus,idiopathic thrombocytopaenic purpura, eosinophilic fasciitis, hyper-IgEsyndrome, antiphospholipid syndrome; other disorders with aninflammatory or immunological component; including acquired immunedeficiency syndrome (AIDS), leprosy, Sezary syndrome, and paraneoplasticsyndromes.

Preferably, the inflammatory disease that can be prevented and/ortreated according to the invention is rheumatoid arthritis.

Antibodies

The term “antibody or antibody fragment” as referred to herein includewhole antibodies and any antigen binding fragment referred to as“antigen-binding portion” or single chains thereof.

An “antibody” refers to a glycoprotein comprising at least two heavy (H)chains and two light (L) chains inter-connected by disulfide bonds, oran antigen binding portion thereof. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as V_(H)) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, C_(H)1 C_(H)2 and C_(H)3. Each light chain is comprisedof a light chain variable region (abbreviated herein as VL) and a lightchain constant region. The light chain constant region is comprised ofone domain, CL. The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each V_(H) and V_(L) is composed of three CDRsand four FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies maymediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (Clq) of the classical complement system.

The term “antigen-binding portion”, as used herein, refers to one ormore fragments of an antibody that retain the ability to specificallybind to an antigen (e.g. BSSL). It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fab'fragment, which is essentially an Fab with part of the hinge region;(iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fvfragment consisting of the VL and VH domains of a single arm of anantibody, (vi) a dAb fragment (Ward et al. 1989) which consists of a VHdomain; (vii) an isolated complementarity determining region (CDR); and(viii) a nanobody, a heavy chain variable region containing a singlevariable domain and two constant domains. Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainFv (scFv); see e.g., Bird et al. (1988). Such single chain antibodiesare also intended to be encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

An “isolated antibody,” as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds BSSL is substantially free of antibodies that specifically bindantigens other than BSSL). An isolated antibody that specifically bindsBSSL may, however, have cross-reactivity to other antigens, such as BSSLmolecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody,” as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences.Furthermore, if the antibody contains a constant region, the constantregion also is derived from human germline immunoglobulin sequences. Thehuman antibodies of the invention may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “human antibody,” as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. In one embodiment, the human monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from atransgenic nonhuman animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

The term “recombinant human antibody,” as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared there from (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(L) and VL sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgGl) that is encoded by the heavy chain constant region genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

The term “human antibody derivatives” refers to any modified form of thehuman antibody, e.g., a conjugate of the antibody and another agent orantibody.

The term “humanized antibody” is intended to refer to antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. Additional framework region modifications may be made withinthe human framework sequences.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

As used herein, an antibody that “specifically binding to human BSSL” isintended to refer to an antibody that binds to human BSSL with a K_(D)of 1×10⁻⁷ M or less, more preferably 5×10⁻⁸M or less, more preferably3×10⁻⁸M or less, more preferably 1×10⁻⁸M or less, even more preferably5×10⁻⁹M or less. The term “does not substantially bind” to a protein orcells, as used herein, means does not bind or does not bind with a highaffinity to the protein or cells, i.e. binds to the protein or cellswith a KD of 1 x 10′ M or more, more preferably 1×10⁻⁵ M or more, morepreferably 1×10⁻⁴ M or more, more preferably 1×10⁻³ M or more, even morepreferably 1×10⁻² M or more.

The term “K_(assoc)” or “K_(a),” as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D),” as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e,. K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A preferred method for determining the K_(D) ofan antibody is by using surface plasmon resonance, preferably using abiosensor system such as a Biacore® system.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) of 1×10⁻⁷ M or less, more preferably 5×10⁻⁸ Mor less, even more preferably 1×10⁻⁸M or less, even more preferably5×10⁻⁹ M or less and even more preferably 1×10⁻⁹ M or less for a targetantigen. However, “high affinity” binding can vary for other antibodyisotypes. For example, “high affinity” binding for an IgM isotype refersto an antibody having a K_(D) of 10⁻⁶ M or less, more preferably 10⁻⁷ Mor less, even more preferably 10⁻⁸ M or less.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows, chickens, amphibians, reptiles, etc.

Anti-BSSL Antibodies

The antibodies to be used according to the invention are characterizedby particular functional features or properties of the antibodies. Forexample, the antibodies bind specifically to human BSSL. Preferably, theantibodies bind to an epitope comprising an amino acid sequence presentin the sequence of human BSSL (SEQ ID NO:2). Most preferably theantibodies bind to an epitope present in the amino acid sequencecorresponding to amino acids 1 to 722 in SEQ ID NO:2, even morepreferably the antibodies bind to an epitope present in the N-terminalpart of BSSL, i.e. an epitope present in the amino acid sequencecorresponding to amino acids 1 to 500 in SEQ ID NO:2.

Preferably, the antibody binds to human BSSL with high affinity, forexample with a K_(D) of 1×10⁷ M or less. The anti-BSSL antibodies to beused according to the invention preferably exhibit one or more of thefollowing characteristics:

(i) binds to human with a K_(D) of 1×10⁻⁷ M or less;

(ii) blocks the binding of to CXCR4 expressing cells;

(iii) blocks enhanced platelet aggregation;

(iv) blocks the binding of BSSL to the complex CXCR4/SDF-1

(v) blocks SDF-1 induced migration of leukocytes

Preferably, the antibody binds to human with a K_(D) of 5×10⁻⁸ M orless, binds to human BSSL with a K_(D) of 2×10⁻⁸ M or less, binds tohuman BSSL with a K_(D) of 5×10⁻⁹ M or less, binds to human BSSL with aK_(D) of 4×10⁻⁹ M or less, binds to human BSSL with a K_(D) of 3×10⁻⁹ Mor less, binds to human BSSL with a K_(D) of 2×10⁻⁹ M or less, or bindsto human BSSL with a K_(D) of 1×10⁻⁹ M or less.

The antibody preferably binds to an antigenic epitope present in humanBSSL, which epitope is not present in other proteins. The antibodytypically binds to human BSSL but does not bind to other proteins, orbinds to other proteins with a low affinity, such as with a K_(D) of1×10⁻⁶ M or more preferably 1×10⁻⁵ M or more, more preferably 1×10⁻⁴ Mor more, more preferably 1×10⁻³ M or more, even more preferably 1×10⁻² Mor more.

Standard assays to evaluate the binding ability of the antibodies towardhuman are known in the art, including for example, ELISAs, Westernblots, RIAs, and flow cytometry analysis. The binding kinetics (e.g.,binding affinity) of the antibodies also can be assessed by standardassays known in the art, such as by Biacore® system analysis.

Production of Monoclonal Antibodies

Monoclonal antibodies (mAbs) to be used according to the presentinvention can be produced by a variety of techniques, includingconventional monoclonal antibody methodology e.g., the standard somaticcell hybridization technique of Kohler and Milstein (1975). Althoughsomatic cell hybridization procedures are preferred, in principle, othertechniques for producing monoclonal antibody can be employed e.g., viralor oncogenic transformation of B lymphocytes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in the mouse is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies to be used according to the presentinvention can be prepared based on the sequence of a non-humanmonoclonal antibody prepared as described above. DNA encoding the heavyand light chain immunoglobulins can be obtained from the non-humanhybridoma of interest and engineered to contain non-murine (e.g., human)immunoglobulin sequences using standard molecular biology techniques.For example, to create a chimeric antibody, murine variable regions canbe linked to human constant regions using methods known in the art (seee.g., U.S. Pat. No. 4,816,567). To create a humanized antibody, murineCDR regions can be inserted into a human framework using methods knownin the art (see e.g., U.S. Pat. No. 5,225,539).

In a preferred embodiment, the antibodies are human monoclonalantibodies. Such human monoclonal antibodies directed against BSSL canbe generated using transgenic or transchromosomic mice carrying parts ofthe human immune system rather than the mouse system. These transgenicand transchromosomic mice include mice referred to herein as the HuMAbMouse® and KM Mouse®), respectively, and are collectively referred toherein as “human Ig mice.”

The HuMAb Mouse® (Medarex®, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg et al.1994). Accordingly, the mice exhibit reduced expression of mouse IgM orκ, and in response to immunization, the introduced human heavy and lightchain transgenes undergo class switching and somatic mutation togenerate high affinity human IgGκ monoclonal antibodies (Lonberg andHuszar 1995). See further, U.S. Pat. No. 5,545,806; and U.S. Pat.No.5,770,429; U.S. Pat. No. 5,545,807; WO 92/03918, WO 93/12227, WO94/25585, WO 97/13852, WO 98/24884, WO 99/45962, and WO 01/14424.

In another embodiment, human antibodies to be used according to theinvention can be raised using a mouse that carries human immunoglobulinsequences on transgenes and transchromosomes, such as a mouse thatcarries a human heavy chain transgene and a human light chaintranschromosome. This mouse is referred to herein as a “KM mouse®,” aredescribed in detail in WO 02/43478.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-antibodies to be used according to the invention. For example, analternative transgenic system referred to as the Xenomouse® (Abgenix,Inc.) can be used; such mice are described in, for example, U.S. Pat.No. 5,939,598; U.S. Pat. No. 6,075,181; U.S. Pat. No. 6,114,598; U.S.Pat. No. 6,150,584 and U.S. Pat. No. 6,162,963. Moreover, alternativetranschromosomic animal systems expressing human immunoglobulin genesare available in the art and can be used to raise anti-BSSL antibodies.For example, mice carrying both a human heavy chain transchromosome anda human light chain transchromosome, referred to as “TC mice” can beused; such mice are described in Tomizuka et al. (2000). Furthermore,cows carrying human heavy and light chain transchromosomes have beendescribed in the art (Kuroiwa et al. 2002) and can be used to raiseanti-BSSL antibodies.

Human monoclonal antibodies which can be used according to the inventioncan also be prepared using phage display methods for screening librariesof human immunoglobulin genes. Such phage display methods for isolatinghuman antibodies are established in the art. See for example: U.S. Pat.No. 5,223,409; U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,571,698; U.S.Pat. No. 5,427,908; U.S. Pat. No. 5,580,717; U.S. Pat. No. 5,969,108;U.S. Pat. No. 6,172,197; U.S. Pat. No. 5,885,793; U.S. Pat. No.6,521,404; U.S. Pat. No. 6,544,731; U.S. Pat. No. 6,555,313; U.S. Pat.No. 6,582,915 and U.S. Pat. No. 6,593,081.

Human monoclonal antibodies can also be prepared using SCID mice intowhich human immune cells have been reconstituted such that a humanantibody response can be generated upon immunization. Such mice aredescribed in, for example, U.S. Pat. No. 5,476,996 and U.S. Pat. No.5,698,767.

RNAi

RNAi molecules that can be used according to the invention comprisesnucleotide sequences complementary to a part of a polynucleotidesequence selected from,

a) the sequence SEQ ID NO:1,

b) a variant of SEQ ID NO:1 having at least 80%, preferably at least90%, such as at least 95%, sequence identity to SEQ ID NO:1, and/or

c) a sequence complementary to the sequences a) and b).

Such RNAi molecules are potential BSSL antagonists.

Antisense

Antisense polynucleotides sequences that can be used according to theinvention comprises nucleotide sequences complementary to a part of apolynucleotide sequence selected from,

a) the sequence SEQ ID NO:1,

b) a variant of SEQ ID NO:1 having at least 80%, preferably at least90%, such as at least 95%, sequence identity to SEQ ID NO:1, and/or

c) a sequence complementary to the sequences a) and b).

Such antisense polynucleotides sequences molecules are potential BSSLantagonists.

The percent sequence identity between two nucleic acid sequences is thenumber of positions in the sequence in which the nucleotide isidentical, taking into account the number of gaps and the length of eachgap, which need to be introduced for optimal alignment of the twosequences.

The percent identity between two polynucleotide sequences is determinedas follows. First, a polynucleotide acid sequence is compared to, forexample, SEQ ID NO:1 using the BLAST 2 Sequences (Bl2seq) program fromthe stand-alone version of BLASTZ containing BLASTN version 2.0.14 andBLASTP version 2.0.14. This stand-alone version of BLASTZ can beobtained from the U.S. Government's National Center for BiotechnologyInformation web site at http://www.ncbi.nlm.nih.gov. Instructionsexplaining how to use the Bl2seq program can be found in the readme fileaccompanying BLASTZ. Bl2seq performs a comparison between twopolynucleotide sequences using the BLASTN algorithm. To compare twopolynucleotide sequences, the options of Bl2seq are set as follows: -iis set to a file containing the first polynucleotide sequence to becompared (e.g., C:\seq1.txt); -j is set to a file containing the secondpolynucleotide sequence to be compared (e.g., C:\seq2.txt); -p is set toblastn; -o is set to any desired file name (e.g., C:\output.txt); andall other options are left at their default setting. For example, thefollowing command can be used to generate an output file containing acomparison between two polynucleotide sequences: C:\Bl2seq-i c:\seq1.txt-j c:\seq2.txt -p blastn -o c:\output.txt. If the two compared sequencesshare sequence similarity, then the designated output file will presentthose regions of similarity as aligned sequences. If the two comparedsequences do not share sequence similarity, then the designated outputfile will not present aligned sequences. Once aligned, the number ofmatches is determined by counting the number of positions where anidentical nucleotide residue is presented in both sequences.

The percent identity is determined by dividing the number of matches bythe length of the sequence set forth in an identified sequence followedby multiplying the resulting value by 100. For example, if apolynucleotide sequence of a length of 120 nucleotides is compared tothe sequence set forth in SEQ ID NO:1 and the sequences once aligned asdescribed above share a sequence where the number of matches is 114,then the sequence has a percent identity of 95% (i.e., 114÷120 * 100=95)to the sequence set forth in SEQ ID NO:1.

BSSL

Briefly, BSSL may be isolated from a suitable tissue such as milk.Alternatively recombinant BSSL can be produced using standard methodsthrough the isolation of DNA encoding BSSL.

DNA encoding BSSL may be conveniently isolated from commerciallyavailable RNA, cDNA libraries, genomic DNA, or genomic DNA librariesusing conventional molecular biology techniques such as libraryscreening and/or Polymerase Chain Reaction (PCR). These techniques areextensively detailed in Molecular Cloning—A Laboratory Manual, 2^(nd)edition, Sambrook, Fritsch & Maniatis, Cold Spring Harbor Press.

The amino acid sequence of human BSSL can be obtained from the SwissProtdatabase, accession no P19835 (CEL_HUMAN) (SEQ ID NO:2) and the cDNAsequence e.g. from the EMBL database accession no. X54457 (SEQ ID NO:1).

The resulting cDNAs encoding BSSL are then cloned into commerciallyavailable mammalian expression vectors such as the pcDNA3 (Invitrogen),pMClneo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo(ATCC 37593), pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC37146), pUCTag (ATCC 37460), 1ZD35 (ATCC 37565), pLXIN, pSIR (Clontech),and pIRES-EGFP (Clontech). Standard transfection technologies are usedto introduce the resulting expression vectors into commonly availablecultured, mammalian cell lines such as L cells L-M(TK—) (ATCC CCL 1.3),L cells L-M (ATCC CCL 1.2), THP-1 (ATCC TIB 202), HEK 293 (ATCC CRL1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650),COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3(ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCCCCL 26) and MRC-5 (ATCC CCL 171). CHO, HEK293, HeLa and clonalderivatives expressing the CEL are isolated. These transfected celllines are used to produce recombinant CEL.

Alternatively the cDNAs encoding BSSL are cloned into commonly availableexpression vectors suitable for expression in micro organisms, such asbacterial expression vectors such as the pET (Invitrogen), pDEST(Invitrogen), pLEX (Invitrogen), pCAL (Stratagene); and the yeastexpression vectors pYES (Invitrogen), pESC (Stratagene) for expressionin saccharomyces and pPICZ (Invitrogen) for expression in pichia.Standard transfection technologies are used to introduce the resultingexpression vectors into commonly available strains of micro organisms,such as the E. coli strains JM101 (Stratagene) and JM110 (Stratagene).

Methods for purification of BSSL from different tissues and transfectedcell-lines are known in the art (Lombardo et al. 1978; Bläckberg andHernell 1981; Wang and Johnson 1983; Hansson et al. 1993).

Formulation and Administration

The antibody and antibody fragments, RNAi molecules and antisensepolynucleotides to be used according to this invention may beadministered in standard manner for the condition that it is desired totreat, for example by oral, topical, parenteral, buccal, nasal, orrectal administration or by inhalation. For these purposes theantibodies and antibody fragments, RNAi molecules and antisensepolynucleotides may be formulated by means known in the art into theform of, for example, tablets, capsules, aqueous or oily solutions,suspensions, emulsions, creams, ointments, gels, nasal sprays,suppositories, finely divided powders or aerosols for inhalation, andfor parenteral use (including intravenous, intramuscular or infusion)sterile aqueous or oily solutions or suspensions or sterile emulsions.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include an anti-BSSL antibody combined with atleast one other anti-inflammatory or immunosuppressant agent.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion).

The pharmaceutical composition of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g. Berge et al. 1977). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, and by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, monthly, once every 3 months or once everythree to 6 months. Preferred dosage regimens for an anti-BSSL antibodyaccording to the invention include 1 mg/kg body weight or 3 mg/kg bodyweight via intravenous, or subcutaneous, administration, or with theantibody being given using one of the following dosing schedules: (i)every four weeks for six dosages, then every three months; (ii) everythree weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg bodyweight every three weeks.

EXAMPLES Example 1 BSSL Appear in the Liver and Co-Localizes withGranulocytes at a State of Liver Steatosis

The hypothesis that the liver could be a source for circulating BSSL wastested.

Subjects and Sample Acquisition

Human liver biopsies were obtained from four patients during electiveabdominal surgery for carcinoma. The biopsies were taken from livertissue at more than one centimeter distant from the site of the tumor.Patient 1 was a 62-year-old man who underwent surgery for colon cancerliver metastasis; patient 2 was a 73-year-old woman who underwentsurgery for rectal cancer liver metastasis; patient 3 was a 60-year-oldwoman who underwent surgery for colon cancer liver metastasis, andpatient 4 was a 63-year-old woman who underwent surgery forcholangiocellular carcinoma. All patients received general anesthesia.

Polymorphonuclear granulocytes and mononuclear cells were isolated fromwhole blood samples from healthy volunteers using the Polymorphprep™(Axis-Shield PoC AS, Oslo, Norway), according to the manufacturer'sguidelines.

Experimental protocols were approved by the Ethics Committee of theMedical Faculty of Umea University, Sweden. Informed consent wasobtained from all participants.

RNA Isolation, cDNA Synthesis, RT-PCR Amplification and Sequencing

Fresh liver specimens collected for RNA isolation were immediatelysubmerged in TRIzol® Reagent (Invitrogen, Carlsbad, Calif., USA) andtotal RNA was isolated according to the manufacturer's instructions.Isolated human blood cells (polymorphonuclear granulocytes andmononuclear cells) were suspended in RNAlater® Solution (Ambion, Austin,Tex., USA) and incubated at 8° C. over night. Cells were pelleted,resuspended in TRIzol®, and total RNA was isolated according to themanufacturer's instructions. The RNA yield was quantifiedspectrophotometrically using a NanoDrop® ND100 (NanoDrop Technologies,Wilmington, Del., USA) and the integrity of the RNA was assessed byethidium bromide staining of ribosomal RNA bands separated on a 1%agarose gel. RNA samples were stored at −70° C. until use.

cDNA was generated from 1 μg of total RNA using random hexamers andTaqMan® reverse transcription reagents in a volume of 100 μl (AppliedBiosystems®, Foster City, Calif., USA).

PCR was performed using AmpliTaq® Gold DNA polymerase (AppliedBiosystems®) according to manufacturer's recommendations. One microliterof cDNA was amplified in a total volume of 20 μl. Primer sequences wereas follows: forward primer (BSSL10) 5′-TCCCGGGACCTGCCCGTTAT-5′(SEQ IDNO:3); reverse primer (BSSL 11) 5′-CTGCAGAGAGACGCTGGCAC-3′ (SEQ IDNO:4). PCR conditions were as follows: 95° C. for 5 min followed by 40cycles of 94° C. for 45 s, 60° C. for 1 min, 72° C. for 1 min, and afinal extension at 72° C. for 8 min. If the target sequence was present,the PCR reaction was expected to produce a 327-bp product, encompassingBSSL exons 4 and 5.

Direct sequencing of PCR fragments was performed using the Big Dye®Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems®) according tomanufacturer's recommendations. BSSL 10 or BSSL 11 (described above) wasused as a primer. The reactions were analyzed using an ABI 3730XL DNAanalyzer (Applied Biosystems®).

Protein Extraction and Western Blot Analysis

Pieces of liver tissue (approximately 100-200 mg) obtained from patients2, 3, and 4 or blood cells (polynuclear granulocytes or mononuclearcells isolated from 10 ml of whole blood) were homogenized in a buffercontaining protease inhibitors [0.047% NH₃, 0.4% Triton™ X-100, 0.08%sodium dodecyl sulfate (SDS), and 1 Mini Complete Tablet per 50 ml(Roche Diagnostics, Mannheim, Germany)]. The homogenate was centrifugedat 14,000 rpm for 10 min and the supernatant was collected and appliedto a HiTrap™ NETS-activated column (GE Healthcare, Buckinghamshire, UK)coupled with anti-human BSSL polyclonal antibodies. The BSSL antibodieswere raised in rabbits and purified as previously described (Hansson etal. 1993). After washing with phosphate buffered saline (PBS)supplemented with 0.02% sodium azide (NaN₃) and 0.01% ethylene diaminetetraacetic acid (EDTA), bound material was eluted by a buffercontaining 0.1 M glycine (pH 2.5), 0.02% NaN₃ and 0.01% EDTA. All stepswere performed at 4° C. to minimize the risk of proteolysis. Elutedproteins were separated on 10% SDS-polyacrylamide gel electrophoresis(PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes(Bio-Rad, Hercules, Calif.). Western blotting was carried out using theECL Advance Western Blotting Detection Kit, following the manufacturer'srecommendations (GE Healthcare). A polyclonal anti-human BSSL antibody(Hansson et al. 1993) was used as primary antibody, and aperoxidase-conjugated donkey-anti-rabbit IgG (DAKO, Glostrup, Denmark)was used as secondary antibody. BSSL isolated from human milk (Bläckbergand Hernell 1981) and protein extracts from human pancreas were used aspositive controls on the western blot.

Histological Analysis and Oil Red O Staining

Specimens for histological evaluation were fixed in 4% paraformaldehyde,0.1 M phosphate buffer (pH 7.0) overnight, embedded in paraffin,microtome-sectioned, and stained with hematoxylin and eosin. For oil red0 staining, tissues were fixed for 2 h at 4° C. in 4% paraformaldehyde,0.1M phosphate buffer (pH 7.0), and cryoprotected by incubation overnight in a solution of 30% sucrose in PBS at 4° C. Thereafter, thespecimens were embedded in Tissue Tek® OCT™ compound (Sakura FinetekEurope B.V., Zoeterwoude, The Netherlands), frozen on dry ice, andstored at −70° C. until sectioning. Upon analysis, 8-μm thick sectionswere cut using a cryostat and mounted on SuperFrost™ Plus slides(Menzel-Gläser, Braunschweig, Germany). Sections were stained with oilred O staining solution (0.3% oil red O in 60% isopropyl alcohol) for 10min at room temperature and then washed with 60% isopropyl alcohol.

Immunohistochemistry and Immunofluorescent Staining

Tissue samples were fixed, embedded, and cryosectioned as describedabove for oil red O staining. Isolated blood cells were applied in adrop of 10 μl onto SuperFrost Plus™ slides (Menzel-Gläser) and allowedto settle for 1 h at room temperature in a humidified chamber. The cellswere washed in 3×PBS (2 min) and 1×PBS (2×2 min) and fixed in 4%paraformaldehyde, 0.1 M phosphate buffer (pH 7.0) for 20 min at roomtemperature.

For single staining-immunohistochemistry, air-dried sections were washedin Tris-buffered saline (TBS; 50 mM Tris-HCl, pH 7.5, 150 mM NaCl) for3×5 min. Endogenous peroxidase activity was blocked by 20 min incubationin a solution of 80% methanol with 0.6% hydrogen peroxide (H₂O₂). Aftersubsequent rinsing in TBS followed by TBS-T (TBS supplemented with 0.1%Triton™ X-100), sections were incubated with 10% normal horse serum(NHS) in TBS-T for 1 h. The first antibody (rabbit anti-BSSL, diluted1:1000 in TBS-T+10% NHS) was applied and incubated for 2 h. Afterwashing in TBS-T (3×5 min), the biotinylated secondary antibody wasapplied [goat anti-rabbit (Vector Laboratories Inc., Burlingame, Calif.,USA), diluted 1:400 in TBS-T+10% NHS] and incubated for 1 h. Sectionswere washed in PBS (3×3 min) and incubated with Vectastain® Elite ABCReagent (Vector Laboratories Inc.) for 1 h, washed again in PBS (3×3min), and developed in diaminobenzidine (DAB) solution [1 tablet of DAB(10 mg) dissolved in 15 ml PBS+12 μl H₂O₂]. Finally, the sections werecounterstained with Mayer's Hematoxylin, dehydrated, and mounted in DPXmicroscopy mounting medium (Merck Sharp & Dohme, Sweden). Negativecontrols comprised sections incubated with rabbit pre-immune seruminstead of the primary antibody.

For immunofluorescence staining, air-dried liver sections or isolatedblood cells, processed and mounted on SuperFrost™ Plus slides as above,were rinsed in PBS for 10 min. Endogenous peroxidase activity wasblocked by incubation in 1% H₂O₂ for 10 min. After washing in PBS (3×3min), sections or cells were incubated with 10% NHS in TBS-T for 1 h.Primary antibodies, diluted in TBS-T+10% NHS, were applied and incubatedfor 2 h. Sections or cells were washed in TBS-T (3×5 min). Secondaryantibodies were applied (diluted 1:1000 in TBS-T+10% NHS), and thesamples were incubated for 1 h. 4′,6-diamidino-2-phenylindole (DAPI;Molecular Probes) was used for nuclear counterstaining. Sections orcells were washed in TBS-T (3×5 min) and mounted with Vectashieldfluorescence medium. Negative controls were composed of sections orcells incubated with rabbit pre-immune serum instead of the primaryantibody. For staining non-permeabilized cells, PBS replaced TBS-T inall steps. The main reactivities for all primary antibodies (apart fromanti-BSSL) are summarized in Table 1.

TABLE 1 Co-localization of immune cell markers and BSSL in human liverCo-localize Marker Main reactivity Dilution with BSSL CD3 Thymocytes, Tcells  1/100 − CD11b Myeloid and NK cells 1/50 + CD14 Myelomonocyticcells  1/100 − CD15 Neutrophils, eosinophils, 1/50 + monocytes CD19 Bcells 1/50 − CD45 All hematopoietic cells  1/100 + CD56 NK cells 1/25 −CD57 NK cells, subsets of T cells,  1/100 − B cells, and monocytes CD68Monocytes, macrophages,  1/100 − neutrophils, basophils, CD86 largelymphocytes 1/50 − Monocytes, activated B cells, dendritic cells HLAclass Antigen presenting cells 1/50 − II DR (B cells, monocytes,dendritic cells, T cells, granulocytes)

The sources and clones were as follows: CD3, clone 289-13801 (MolecularProbes, Eugene, Oreg., USA); CD11b, clone 2LPM19C (DacoCytomation,Glostrup, Denmark); CD14, clone TÜK4 (DacoCytomation); CD15, clone C3D-1(DacoCytomation); CD19, clone HD37 (DacoCytomation); CD45, clone HI30(BD Biosciences, San Jose, Calif., USA); CD56, clone T199(DacoCytomation); CD57, clone NC1 (Immunotech, Marseilles, France);CD68, clone KP1 (DacoCytomation); CD86, clone FUN-1 (BD Biosciences);HLA class II DR, clone CR3/43 (DacoCytomation). The secondary antibodiesused were Alexa fluor® 488 goat-anti-rabbit, Alexa fluor® 488goat-anti-mouse, Alexa fluor® 594 goat-ant-rabbit, and Alexa fluor® 594goat-anti-mouse (Molecular Probes).

Results BSSL is Expressed in Human Liver Biopsies

Total RNA was extracted in duplicate from human liver biopsies collectedfrom four patients (nos. 1-4). The RNA was reverse transcribed andamplified using BSSL-specific oligonucleotide primers designed to targetexons 4-5. A PCR product corresponding to the expected size (327 nt) wasamplified from all samples (FIG. 1). The 327-nt PCR fragments weresequenced and found to be identical to the published human BSSL cDNAsequence (EMBL accession no. X54457; data not shown).

Protein extracts were prepared from liver biopsies from patient no. 3and no. 4 and applied to an anti-BSSL-sepharose column. After washing,the bound material was eluted and subjected to western analysis. Asingle protein with a molecular mass corresponding to the mass of humanmilk BSSL was detected in both samples (FIG. 2). The molecular mass ofBSSL in the liver was comparable to that of BSSL found in human milk butslightly greater than the mass of the BSSL found in human pancreas.

Immunohistochemistry Localizes BSSL to Polynuclear Granulocytes in HumanLiver

Hematoxylin-eosin and oil red O-staining of liver sections revealed thatpatient no. 4 suffered from extensive liver steatosis (FIGS. 3D and 3E).In contrast to patient no. 1 (FIGS. 3A and B), the entire section frompatient no. 4 was crowded with large lipid-filled vacuoles.Immunohistochemistry using BSSL-specific antibodies on liver sectionsderived from patients 1 and 4 confirmed the presence of BSSL in humanliver (FIGS. 3C and 3F). In sections from patient no. 4, cells thatstained positive for BSSL seemed to cluster around the large lipiddroplets (FIG. 3F), and the number of BSSL-positive cells was at least10-fold higher in patient no. 4 than in patient no. 1. Moreover, cellsthat stained positive for BSSL in patient no. 1 did not cluster but wereevenly scattered throughout the entire section (FIG. 3C). TheBSSL-positive cells did not resemble hepatocytes morphologically, butinstead resembled stellate cells or immune cells.

To investigate which cell type(s) expressed BSSL in human liver, doubleimmunofluorescence staining was performed on tissue sections derivedfrom patients no. 1 and no. 4. No co-localization was found between BSSLantibodies and antibodies directed toward smooth muscle actin ordesmine, two antigens present on stellate cells (data not shown). Incontrast, BSSL antibodies clearly co-localized with antibodies directedtoward the leukocyte common antigen CD45, confirming that localized toimmune cells (data not shown). To further investigate which cellsexpressed BSSL, we examined whether co-localized with different antigenspresent on a variety of immune cells (Table 1). Antibodies against CD3,CD14, CD19, CD56, CD57, CD86, and HLA class II DR all failed toco-localize with BSSL antibodies (data not shown), as did antibodiesagainst CD68 (FIGS. 4A and 4B). However, anti-CD15 antibodies (presenton 95% of mature granulocytes) and CD11b (present on myeloid cells andNK cells) clearly co-localized with BSSL-expressing cells (FIGS. 4A and4B (CD15) and data not shown (CD11b)). These data showed that BSSL inhuman liver was not expressed by hepatocytes or other liver-specificcells, nor by macrophages as previously proposed, but most likely bygranulocytes.

BSSL is Expressed by Circulating Blood Cells

Immunofluorescence studies revealed that BSSL and CD15 co-localized inpermeabilized polymorphonuclear leukocytes isolated from whole humanblood (FIG. 5). In contrast, anti-BSSL antibodies did not react toCD14-positive mononuclear cells (data not shown). Hence, in thecirculation, BSSL was expressed by, or at least associated with,polymorphonuclear granulocytes. When immunofluorescence staining wasperformed on permeabilized and non-permeabilized granulocytes,BSSL-positive staining occurred only in permeabilized granulocytes (FIG.6). In contrast, CD15 antibodies stained both permeabilized andnon-permeabilized cells.

Polynuclear granulocytes and mononuclear cells were isolated separatelyfrom human blood. Protein extracts were generated from each cellpopulation and applied to an anti-BSSL-sepharose column. Bound andeluted material was resolved by western immunoanalysis. Polyclonalanti-BSSL antibodies detected a single protein with a molecular masscorresponding to the mass of human milk in both polynuclear granulocytesand mononuclear cells (FIG. 7).

Total RNA isolated from polynuclear granulocytes and mononuclear bloodcells was analyzed for the presence of BSSL mRNA by RT-PCR. A PCRproduct of the expected size (327 nt) was generated from both cellfractions (FIG. 8). Direct sequencing of the PCR fragments revealed asequence identical to that of the published human BSSL cDNA (EMBLaccession no. X54457; data not shown).

Example 2 BSSL is Present in Atherosclerotic Plaque HistologicalAnalysis and Immunohistochemistry

Specimens of human atherosclerotic carotid artery were fixed in 4%paraformaldehyde, 0.1 M phosphate buffer (pH 7.0) overnight, embedded inparaffin and microtome-sectioned. Immunohistochemistry was performed asdescribed above. A polyclonal rabbit anti-human BSSL (directed againstamino acid 328-341) was used as primary antibody in these experiments.

Results

The presence of BSSL in human atherosclerotic plaque was confirmed (FIG.9). Taken together, the data presented above (Examples 1 and 2) suggestthat BSSL, in addition to being a key enzyme in dietary fat digestion inearly life, is also involved in inflammatory processes such as liversteatosis and atherosclerosis.

Example 3 BSSL Deficient Mice are Protected from Collagen InducedArthritis (CIA)

Following the demonstration that BSSL is produced by granulocytes andplatelets and present at the site of inflammation (liver steatosis andatherosclerotic plaques), the hypothesis that BSSL is involved invarious conditions with inflammation as a common denominator, e.g.autoimmune arthritis was tested.

For this purpose the response of BSSL deficient “knockout” (BSSL-KO)mice was compared to wild-type mice in a collagen-induced arthritis(CIA) model (Courtenay et al. 1980). CIA is a commonly used experimentalmodel in mice and rats that reproduces many of the pathogenic mechanismsof human rheumatoid arthritis (RA), i.e. increased cellularinfiltration, synovial hyperplasia, pannus formation and erosion ofcartilage and bone in the distal joints.

Study Design

BSSL-KO and BSSL-WT mice were immunized with collagen type II (CII) incomplete Freunds adjuvant (CFA) day 0 and boosted with collagen type II(CII) in incomplete Freunds adjuvant (IFA) day 21, according to standardprotocol. Severity of disease was followed for 57 days. Blood was takenday 30 and at the end of experiment (day 57).

Mice

To obtain susceptibility to CIA, conferred by the MHC A^(q) haplotype,BSSL-KO mice of C57BL/6 background (gift from Dr. J. Breslow,Rockefeller University, New York) were crossed to the C57BL/10Qbackground for one generation (F1). heterozygous mice were theninter-crossed to generate BSSL-KO and BSSL-WT littermates, all carryingthe MHC A^(q) allele. These littermates were employed for theexperiment.

Procedures

33 Males and 32 females from intercross generation F1 were used. Themice were bred and kept at 12 h light/dark cycles, in polystyrene cagescontaining wood shavings and were fed with standard rodent chow andwater ad libitum at the animal house Umeå University. All mice includedwere either homozygous (n=26) or heterozygous (n=39) for the MHC A^(q)haplotype allowing CII responsiveness (Wooley et al. 1981). In total 37BSSL knock out (ko) and 28 wild type (wt) littermate mice were includedin the experiment.

Mice were immunized with 100 μg rat CII in CFA, total volume of 50 μl atthe base of the tail day 0. Emulgate was prepared in syringes using aconnector (black) and kept on ice until use. A booster injection wasperformed day 21 with 50 μg rat CII in IFA (total volume 50 μl). Blindedclinical scoring of CIA was performed using a system based on the numberof inflamed joints in each mouse. Inflammation was defined by theswelling and redness of the joints. Blood was taken by cheekbleeding day30 and at the end of the experiment (day 57). The blood was taken inheparinised tubes and centrifuged to separate plasma (4,000 rpm, 10min). Plasma was stored at −20° C. until assayed.

Plasma concentration of cartilage oligomeric matrix protein (COMP) wasdetermined by a competitive ELISA according to an earlier describedmethod (Saxne et al. 1992). Briefly, rat COMP was used for coating ofthe microtiter plates and for preparing the standard curve included ineach plate. Plates were blocked with 1% bovine serum albumin (BSA) inPBS for 2 hours in room temperature. After blocking, plasma co-incubatedwith rabbit polyclonal antiserum against rat COMP (generously providedby Professor Dick Heinegård, Lund, Sweden) was added and the plates wereincubated for 2 hours at room temperature. The amount of plasma COMP wasestimated after incubation with an alkaline phosphatase-conjugatedswine-anti-rabbit isotype-specific antibody (DAKO, Glostrup, Denmark)and phosphatase substrate (Sigma Aldrich) as substrate followed bydetection in a Spectra Max® (Molecular Devices, Sunnyvale, Calif., USA)at OD 405 nm.

The antibody response against rat CII in plasma was determined withELISA in 96-well plates (Costar, Cambridge, Mass. USA) coated overnightat 4° C. with 50 μl well of 10 μg/ml rat CII in 50 μl PBS. All washeswere performed with PBS (pH 7.4) containing 0.1% Tween®-20. Plasma wasdiluted in PBS and analyzed in duplicates. The amounts of bound IgGantibodies were estimated after incubation with biotin-conjugatedisotype-specific antibodies (Southern Biotechnology Associates, Inc.Birmingham, Ala., USA) followed by Extravidin®-Peroxidase (Sigma) anddeveloped with ABTS (Roche Diagnostics GmbH, Mannheim, Germany) assubstrate followed by detection in a Spectra Max® at OD 405 nm(Molecular Devices).

Results

The results from the CIA experiment (FIGS. 10-13) show a significantprotection from disease in mice that are knocked out for the BSSL gene.BSSL-KO mice develop arthritis with less incidence and lower severity(FIG. 11). The effect was mainly seen in males, but it is difficult todraw a conclusion on sex specificity since the females developedarthritis with too low incidence in general, and the disease developedwith some delay relative to males. This was not surprising, since it iswell known that male mice are more often affected than females in theCIA model. There was no difference in antibody response against CII(FIG. 13) but significantly less cartilage degradation in BSSL-KO micewhich correlates with the arthritis development (FIG. 13).

Example 4 Collagen Induced Arthritis in BSSL-Deficient Mice (Follow-Up)

The CIA experiment described above was repeated with the same protocoland end-points (clinical scoring, anti-CII antibody response and COMPplasma concentration), but for this second CIA experiment BSSLheterozygote (BSSL-HET) mice were included and the study was limited tomale mice. This follow-up study confirmed the results above and furthershowed that BSSL-HET mice were less prone to develop disease as comparedto BSSL-WT mice but not as resistant as BSSL-KO mice (FIGS. 14-16).

Example 5 Pristane Induced Arthritis in Rats

It was hypothesized that antibodies directed towards could preventbinding of BSSL to its target and hence serve as therapeutic agents toblock and/or ameliorate arthritis severity. To test this hypothesis invivo, the effect of anti-BSSL antibodies was investigated in anotheranimal model of autoimmune arthritis, i.e. pristane-induced arthritis(PIA) in rats.

Study Design

Dark Agouti (DA) rats, known to have a high susceptbility for developingPIA, were injected with pristane at day 0. At day 5, 10 and 15 the ratswere injected with one of the following; 1) PBS, 2) anti-BSSL 1 mg/kg or3) anti-BSSL 5 mg/kg) (n=10 for each group). Development of disease(arthritis severity) was followed by clinical scoring as described forthe CIA model above.

Rats

male DA rats from Harlan Laboratories, Boxmeer, The Netherlands (8-10weeks at arrival) were kept at 12 h light/dark cycles in polystyrenecages containing wood shavings and were fed with standard rodent chowand water ad libitum at the conventional animal house of BMC, LundUniversity, Lund. The experiment was approved by the Malmö/Lund ethicalcommittee' under license number M107-07. One rat died during anesthesiaduring the experiment and was excluded. The rats were anesthetized forall injections.

Procedures

PIA was induced by s.c. injection at the base of the tail with 150 μlpristane day 0 using a 0.6×25 mm needle. Day 5, 10 and 15 rats wereinjected with either of the following treatments intraperitoneally(i.p.) in a total volume of 1 ml/rat a) PBS, b) polyclonal rabbitanti-human BSSL antibody (directed against amino acid 328-341) 1 mg/kgor c) anti-BSSL antibody 5 mg/kg (n=10 for each group). The rats wereevaluated for arthritis severity from day 9 and until the end ofexperiment (day 22).

At the end of experiment, paws from representative rats were collectedand fixed in 4% PFA, alternatively put in decalcifying EDTA solution.Fixed samples were moved to EDTA solution after 24 hours.

Results

The results from the PIA experiment showed that anti-BSSL antibodies (5mg/kg) significantly reduced disease severity when injected at theinitiation of disease (FIG. 17). Even in the group injected with thelower dose (1 mg/kg) a tendency towards amelioration was found.

Conclusions BSSL in Inflammatory Diseases

These present data demonstrate that BSSL, in addition to being a keyenzyme in dietary fat digestion in early life, is present ingranulocytes and involved in inflammatory processes. The present datafurther demonstrate that there is a requirement for BSSL in theinflammatory process and response in inflammatory diseases. Lack of BSSLor treatment with antibodies directed to BSSL significantly reduceddisease severity in two animal models of rheumatoid arthritis.

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1. (canceled)
 2. A method for inhibiting leukocyte migration in asubject suffering from ulcerative colitis or Crohn's disease, the methodcomprising administering a pharmaceutical effective amount of anantibody that binds specifically to human bile salt-stimulated lipase(BSSL) to the subject in absence of another immunosuppressive agent,wherein the antibody binds to an epitope present in the N-terminal partof human BSSL corresponding to amino acids 1 to 500 in SEQ ID NO: 2; andthe antibody inhibits, when bound to human BSSL, leukocyte migration. 3.The method according to claim 2, wherein the antibody is a monoclonalantibody.
 4. The method according to claim 2, wherein the subjectsuffers from ulcerative colitis.
 5. The method according to claim 2,wherein the subject suffers from Crohn's disease.
 6. The methodaccording to claim 2, wherein the antibody binds to an epitopecorresponding to amino acid numbers 328 to 341 in human BSSL.
 7. Amethod for treating ulcerative colitis or Crohn's disease in a subject,the method comprising administering a pharmaceutical effective amount ofan antibody that binds specifically to human bile salt-stimulated lipase(BSSL) to the subject in absence of another immunosuppressive agent,wherein the antibody binds to an epitope present in the N-terminal partof human BSSL corresponding to amino acids 1 to 500 in SEQ ID NO: 2; theantibody inhibits, when bound to human BSSL, leukocyte migration; andinhibition of leukocyte migration treats ulcerative colitis or Crohn'sdisease in the subj ect.
 8. The method according to claim 7, wherein theantibody inhibits, when bound to human BSSL, binding of human BSSL toCXCR4 and thereby blocks CXCR4 binding to SDF-1 and inhibits SDF-1induced leukocyte migration.
 9. The method according to claim 7, whereinthe antibody is a monoclonal antibody.
 10. The method according to claim7, wherein the subject suffers from ulcerative colitis.
 11. The methodaccording to claim 7, wherein the subject suffers from Crohn's disease.12. The method according to claim 7, wherein the antibody binds to anepitope corresponding to amino acid numbers 328 to 341 in human BSSL.